Quantitative clotting assay for activated factor VII

ABSTRACT

An assay for activated factor VII (factor VIIa) has been developed using truncated tissue factor (tTF), a soluble mutant form of tissue factor (TF) that retains the cofactor function of TF toward factor VIIa. Unlike full-length TF, however, tTF appears not to support the conversion of factor VII to VIIa. As a result, the tTF assay for factor VIIa is free from interference from factor VII in the plasma and is therefore specific for factor VIIa. The assay is much simpler than existing assays, because it is a single-stage clotting assay performed almost identically to a prothrombin time (PT) assay. It is also considerably more sensitive than current assays for factor VIIa in plasma. Since the tTF assay is calibrated against a factor VIIa standard, it yields an absolute concentration of factor VIIa in ng/ml.

The United States government has rights in this invention by virtue of agrant from the National Institutes of Health.

RELATED APPLICATION DATA

This application is a continuation of U.S. patent application Ser. No.07/683,682, filed Apr. 10, 1991 now U.S. Pat. No. 5,472,850.

TECHNICAL FIELD

This invention relates to the field of detection of blood factorsinvolved in blood coagulation.

BACKGROUND OF THE INVENTION

Blood coagulation results from the production of thrombin, a proteolyticenzyme inducing platelet aggregation and cleaving fibrinogen to fibrin,which stabilizes the platelet plug. A number of proenzymes andprocofactors circulating in the blood interact in this process throughseveral stages during which they are sequentially or simultaneouslyconverted to the activated form, ultimately resulting in the activationof prothrombin to thrombin by activated factor X (fXa) in the presenceof factor Va, ionic calcium, and platelets.

Factor X can be activated by either of two pathways, termed theextrinsic and intrinsic pathways. The intrinsic pathway, orsurface-mediated activation pathway, consists of a series of reactionswhere a protein precursor is cleaved to form an active protease,beginning with activation of factor XII to factor XIIa, which convertsfactor XI to factor XIa, which, in the presence of calcium, convertsfactor IX to factor IXa. Factor IX can also be activated via theextrinsic pathway by tissue factor (TF) in combination with activatedfactor VII (factor VIIa; fVIIa). The activated factor IX, in thepresence of calcium, phospholipid (platelets), and factor VIIIa,activates factor X to factor Xa.

Physiologically, the major pathway involved in coagulation is believedto be the extrinsic pathway, an essential step of which is activation offactor VII to factor VIIa. Clotting assays (and other activity assays)designed to measure factor VII and VIIa generally must employ TF, thecofactor required for factor VIIa coagulant activity. Most commonly, TFis provided as a relatively crude preparation known as thromboplastin.Tissue factor is an integral membrane glycoprotein having a protein anda phospholipid component. It has been isolated from a variety of tissuesand species and reported to have a molecular mass of between 42,000 and53,000. The original crude tissue abstracts referred to the fractioncontaining the tissue factor as thromboplastin. DNA encoding tissuefactor and methods for expression of the protein have now been reported,for example, in European Patent Application 0 278 776 (3/1988) byGenentech, Inc. and by J. H. Morrissey, et al., "Molecular cloning ofthe cDNA for tissue factor, the cellular receptor for the initiation ofthe coagulation protease cascade," Cell 50:129-135 (1987).

The complex of factor VIIa and TF is the most potent known trigger ofthe clotting cascade. Factor VII is present in plasma at a concentrationof 0.5 μg/ml plasma. In contrast, measured as described herein, factorVIIa is present in plasma at trace levels of roughly 1 ng/ml.Accordingly, factor VII is normally in considerable excess over factorVIIa. As there are presently no immunoassays capable of discriminatingbetween factor VII and VIIa, current assays attempt to exploit thedifference in enzymatic activity between the two, relative to totalfactor VII protein.

Factor VIIa circulates with a relatively long half-life of about twohours in plasma. This is an unusual property among activated coagulationenzymes, which typically are inactivated very rapidly by variousprotease inhibitors in plasma. Some studies have suggested that elevatedlevels of plasma factor VIIa are associated with increased risk ofthromboembolic disease. This may be due to a direct effect of factorVIIa in promoting thrombosis, or because elevated plasma factor VIIa isa by-product of a "prethrombotic stated". In either case, there isclinical interest in measuring plasma factor VIIa levels.

Two basic types of assays are routinely employed to assess coagulationfunction: clotting assays and chromogenic substrate assays. Clottingassays compare clotting times relative to normal controls (time inseconds to clot formation versus percent normal control, also referredto as one unit). Chromogenic substrate assays are typically used onlywith the purified enzymes and the amount of active factor calculatedbased on the amount of product generated in a given time as determinedby absorbance of light transmitted through the sample at a particularwavelength. Immunological assays involving antigen-antibody reactionsare also sometimes used to measure the total amount of protein present.

Prothrombin time (PT) and activated partial thromboplastin time (aPTT)assays are routinely employed clinically in the assessment of hemostaticfunction. PT is performed by adding a preparation of phospholipid,tissue factor and calcium to the patient's citrated plasma anddetermining the time required for a clot to form. This test measures theaggregate activity of factors II, VII, X, V and fibrinogen. The PT isoften used to follow oral anticoagulant therapies, i.e., coumarin andanti-vitamin K drugs. The aPTT test is performed by adding a preparationof celite (or other contact activator), phospholipid, and calcium to thepatient's citrated plasma and determining the time required for a clotto form. This test measures the aggregate activity of factors XII, XI,IX, VIII, X, II, V and fibrinogen. The aPTT is commonly used as acoagulation screening test for factor dysfunction in hospitalizedpatients.

In contrast to these general assays, quantitative assays of specificindividual coagulation factors are usually performed using a plasmadeficient in the factor to be assayed and comparing clotting timesrelative to controls. Typically, the amount of factor VII/factor VIIapresent in a patient's plasma would be determined using factor VIIdeficient plasma, adding a dilution of patient plasma, TF, and calcium,usually in the form of calcium chloride, and determining the timerequired for the plasma mixture to clot. The result is a measure of whatis called "factor VII coagulant activity", or FVIIc. In reality, FVIIcis a function of the concentrations of both factor VII and VIIa. It isdifficult to assess the amount of factor VII relative to the amount offactor VIIa, however, since TF greatly accelerates the activation offactor VII, and the newly converted factor VIIa contributes to themeasured activity. A further complication of the significance of FVIIcis the recent finding of an unusual, lipoprotein-associated form offactor VII that has elevated FVIIc activity. The nature of thislipoprotein-associated form of factor VII is poorly understood at thepresent time.

These factors limit the sensitivity of existing assays and complicatetheir interpretation. In conventional clotting assays, human factor VIIahas approximately 25-fold more activity than factor VII on an equimolarbasis. This means that prior assays have had difficulty detectingconcentrations of factor VIIa protein that are much below 1/25 of theconcentration of factor VII. The normal plasma concentration of factorVII is approximately 0.5 μg/ml, and the lower limit of detection offactor VIIa in normal plasma using the conventional factor VII clottingassay is between 1 and 10 ng/ml.

In order to try to control for possible variation of plasma factor VIIlevels, another assay is usually performed in parallel that is designedto measure total factor VII plus VIIa. This can be an immunoassay or achromogenic substrate assay in which factor VII is converted to factorVIIa. The final result is then expressed as a ratio of factor VIIclotting activity (FVIIc) to total factor VII, and an increase in theratio above normal is taken as evidence of an increased level of factorVIIa in the test plasma. These assays do not really measure factor VIIadirectly and specifically, nonetheless, nor do these assays give ameasure of absolute level of factor VIIa. It is thought that elevatedFVIIc can be due to the presence of elevated circulating factor VIIalevels. Alternatively, elevated FVIIc can be due to an increasedconcentration of factor VII in plasma. In any case, the results fromconventional assays of FVIIc depend heavily on the source ofthromboplastin, as reported by Poggio, et al., "Factor VII clottingassay: influence of different thromboplastins and Factor VII-deficientplasmas," Thrombosis Haemostasis, 65:160-164 (1991), making it difficultto compare the results of such assays from one laboratory with another.

In summary, prior art methods for measuring factor VIIa levels in plasmaare limited by the interference of factor VII which is also present inplasma. There are therefore three main drawbacks to the availableassays: (1) it is extremely difficult to measure absolute levels offactor VIIa in plasma, as compared with levels of factor VII; (2) lowsensitivity of available assays means that only factor VIIa levelssubstantially increased over normal can be measured; and (3) it isdifficult to directly compare the results obtained in different studies.

It is therefore an object of the present invention to provide asensitive assay which measures the absolute levels of factor VIIa, notfactor VII, in plasma.

It is further object of the present invention to provide an assay forfactor VIIa which is reproducible and commercially viable and can beperformed with existing methodologies and most reagents, manually orautomatically.

SUMMARY OF THE INVENTION

An in vitro assay for detecting and quantitating factor VIIa (theactivated form of coagulation factor VII) in human plasma has beendeveloped. The single-stage clotting assay is performed in a mannernearly identical to the prothrombin time (PT) clotting assay using asmall sample of blood drawn into a citrate anticoagulant, from whichplasma is prepared by centrifugation. A modified clotting assay is thenperformed on a sample of the plasma using a preparation of truncatedtissue factor and phospholipid vesicles in place of thromboplastin. Thetruncated derivative of tissue factor retains cofactor activity towardfactor VIIa, but, unlike normal tissue factor, does not stimulate theconversion of factor VII to factor VIIa. As a result, unactivated factorVII, which is also present in plasma, does not interfere with the assay.The clotting time obtained in this assay is directly related to thelevel of plasma factor VIIa and is quantitated by comparison to astandard curve generated using factor-VII deficient plasma supplementedwith known quantities of factor VIIa. The useful range of the assay is10 pg factor VIIa/ml to 10 μg factor VIIa/ml in plasma. The lower limitof detection of factor VIIa in the truncated tissue factor-based assayis calculated to be less than 10 pg factor VIIa/ml.

The assay is useful in accurately measuring plasma levels of factor VIIain individuals. Recent studies have indicated a possible correlationbetween elevated factor VIIa levels and the risk of such disorders asischemic heart disease and fetal growth retardation. The assay can alsobe used to monitor factor VIIa therapy in hemophiliacs and otherindividuals undergoing factor VIIa therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the amino acid and nucleotide sequence encoding a soluble formof truncated tissue factor. Nucleotide sequence is numbered on the left.Amino acid sequence is given above the nucleotide sequence using thestandard one-letter code. The signal peptide is underlined.

FIG. 2 is an exemplary factor VIIa standard curve generated using anautomated coagulometer (ACL™ 300 Automated coagulation Laboratory) runin the PT mode, using a high-sensitivity tTF reagent. Human factorVII-deficient plasma was supplemented with varying concentrations ofrecombinant human factor VIIa spanning the anticipated normal range ofFVIIa concentrations. The tTF reagent contained 1 μM tTF. Data (clottingtimes vs. added factor VIIa concentration) were plotted on log-log axesand fit to a fourth order polynomial by regression analysis.

FIG. 3 is an exemplary plot of clotting times obtained in the tTF assayfor factor VIIa with a low-sensitivity tTF reagent and normal humanplasma supplemented with factor VIIa at levels that mimic thetherapeutic range. The tTF reagent contained 50 nM tTF. Data (clottingtimes vs. added factor VIIa concentration) were plotted on log-log axesand fit to a fourth order polynomial by regression analysis.

FIG. 4 is an exemplary factor VIIa standard curve generated by themanual tilt tube method and employing a high-sensitivity tTF reagent.Human factor VII-deficient plasma was supplemented with varyingconcentrations of recombinant human factor VIIa (spanning 10 pg/ml to 10μg/ml fVIIa). The tTF reagent contained 100 nM tTF. Data (clotting timesvs. added factor VIIa concentration) were plotted on log-log axes andfit to a fourth order polynomial by regression analysis.

DETAILED DESCRIPTION OF THE INVENTION

Clotting of citrate-anticoagulated, platelet-poor plasma is initiated bya combination of truncated tissue factor, phospholipid vesicles andcalcium ions. The clotting time is an indicator of the level of factorVIIa present in the plasma.

There are two preferred embodiments of the assay for activated factorVII: a manual version, using 100 nM tTF and a relatively lowconcentration of phospholipids, and an assay for use in automatedcoagulometers and fibrometers, which most clinical labs in the US areusing. The manual assay can be manipulated to have a million-fold rangein sensitivity, ranging from 10 pg/ml to 10 μg/ml. This is not practicalusing the automated coagulometers due to small but significant reagentcarry-over. Accordingly, two "stock" solutions of the tTF reagent,containing different amounts of tTF and/or phospholipid, are maintained:one, for measuring the normal range of fVIIa levels, approximately 0.1to 10 ng/ml, and a second for measuring the therapeutic range of fVIIalevels, up to about 4 μg/ml.

Reagents for Use in Activated Factor VII Assays

The reagents common to all embodiments of the assay are the plasmasample, a source of phospholipid, a source of clotting factors otherthan factor VIIa, truncated tissue factor (tTF), and calcium.

1. Phospholipid Vesicles

In the preferred embodiment, the formulation consists of 100 nMtruncated tissue factor reagent (2.55 μg/ml) plus 80 μg mixed brainphospholipid vesicles/ml suspended in TBS/BSA (50 mM Tris.HCl pH 7.4,100 mM NaCl, 0.02% sodium azide, 0.1% bovine serum albumin).Phospholipid vesicles (600 μM) can be prepared by the deoxycholatemethod of Carson and Konigsberg, Thrombosis Haemostasis 44:12-15 (1981).Phospholipid vesicles are not absolutely required, but their additionincreases the sensitivity of the assay. It is believed that the presenceof the vesicles decreases variability in the assay, since coagulation isless dependent on the residual content of lipoprotein particles,platelets and cell membrane fragments in the plasma samples.

Cephalin, prepared according to Bell and Alton, Nature, London,174:880-881 (1954), the teachings of which are incorporated herein, andavailable commercially from a number of suppliers, including, forexample, Rabbit Brain Cephalin from Sigma Chemical Company, St. Louis,Mo., can also be used as the source of phospholipid vesicles. Thevesicle preparations are reconstituted according to the manufacturer'sinstructions and employed at the final concentration recommended for usein the partial thromboplastin time (PTT) assay, described by Langdell,et al., J Lab Clin Med 41:637 (1953).

Other preparations of phospholipid vesicles suitable for use as a"platelet substitute" in PTT assays or thrombin generation time (TGT)assays are acceptable for use in the truncated TF assay for factor VIIa,including vesicles prepared from crude phospholipid extracts of tissuesas well as vesicles prepared from purified or synthetic phospholipids.Such vesicle preparations must not contain tissue factor or additivesthat activate the contact pathway of coagulation. Examples of unsuitablepreparations of vesicles are commercial thromboplastins designed for usein PT assays and "activated partial thromboplastins", cephalinpreparations admixed with activating agents such as elegiac acid,kaolin, or venom extracts.

2. The tTF Reagent

As used herein, "truncated tissue factor" is a soluble tissue factorhaving the extracellular domain which is not bound to a phospholipidmembrane surface, and therefore does not support conversion of fVII tofVIIa. In the preferred embodiment, truncated tissue factor is arecombinant protein produced in vitro in cell culture using a human cellline (293 cells), ATCC CRL 1573. These cells are stably transfected witha mutant form of the human tissue factor cDNA carried in a commerciallyavailable expression vector, and secrete a form of tissue factorconsisting only of amino acids 1-219 (numbered according to Morrissey,et al., Cell 50:129-135 (1987), the teachings of which are incorporatedherein).

The recombinant, truncated tissue factor is purified from the culturemedium using an immobilized monoclonal antibody to human tissue factor,such as TF9-5B7, described in Morrissey, et al., "Monoclonal antibodyanalysis of purified and cell-associated tissue factor," ThrombosisResearch 52:247-261 (1988). Hybridomas for production of monoclonalantibodies can be propagated by ascites growth and the monoclonalantibodies (MAbs) purified from ascites fluid using the BioRad MAPS IIsystem for MAb purification, as described by Morrissey, et al.,Thrombosis Research 52:247-261 (1988).

The TF9-5B7 is coupled to Affigel™ beads. Detergent is not used duringthe purification of TF₂₁₉. After removal of cellular debris bycentrifugation, the culture medium is made 25 mM in Tris.HCl (pH 7.4),10 mM in sodium EDTA (pH 7.4), and 0.1% in sodium azide by the additionof concentrated stock solutions. In order to remove proteins that bindto agarose beads non-specifically, the culture medium is gently agitatedfor 4 hr at 4° C. with AffiGel™ -10 beads that had previously beenblocked chemically with glycine ethyl ester (GEE-AffiGel™). TheGEE-AffiGel™ beads are removed by filtration through a sintered glassfunnel, and the supernatant is agitated overnight at 4° C. with the MAbTF9-5B7 coupled to AffiGel™ beads (typically 2 ml of beads). TheTF9-5B7-AffiGel™ beads are collected on a sintered glass funnel, and thebeads are washed on the funnel with 100 ml of TBS-EDTA (TBS=100 mM NaCl,50 mM Tris.HCl pH 7.4, 0.02% sodium azide; TBS-EDTA=TBS with 10 mM EDTAincluded). The beads are then transferred to a chromatography column andwashed with 40 ml TBS followed by 40 ml of a solution consisting of 1MNaCl, 10 mM Tris.HCl pH 7.4, 0.02% sodium azide. Truncated TF is elutedfrom the beads using 100 mM glycine.HCl pH 2.5, with 1 ml fractionsbeing collected into tubes containing 57 μl 1M Tris base (to immediatelyneutralize the acidic glycine buffer). Fractions containing protein aredetected using the BCA protein assay (Pierce), pooled, dialyzed againstTBS, and then stored at -70° C. Protein concentrations are determinedusing standard methods such as the BCA assay (Pierce Chemical Co.) basedon a bovine serum albumin standard of known concentration.

The TF₁₋₂₁₉ deletion mutant used in the preferred embodiment wasconstructed and stably expressed in eukaryotic cells as follows. Adeletion mutant of human TF cDNA was created that contained the codingsequence for the predicted leader peptide and extracellular domains, butlacked the predicted transmembrane and cytoplasmic domains. Startingwith clone pCTF543 (described by Mackman et al., Biochemistry28:1755-1762 (1989)), a 775 bp fragment of TF cDNA was released bydigestion with EcoRI. This fragment extended from the 5' noncodingsequence to the EcoRI site in the coding sequence (nucleotides 1 through775 as numbered according to Morrissey et al., Cell 50:129-135 (1987)).To each end of the 775 bp cDNA fragment was ligated a double-strandedsynthetic oligonucleotide made by annealing together the following twosingle-stranded oligonucleotides (sequences listed 5' to 3'):AATTTAGAGAATAAGAATTCGGG (SEQ ID NO: 1) and CCCGAATTCTTATTCTCTA (SEQ IDNO: 2). On the 3' end of the insert, the adaptor took advantage ofredundancy in the codon for phenylalanine by changing the codon forPhe²¹⁷ from TTC to TTT, thereby destroying the original EcoRI site butpreserving the encoded amino acid sequence through Glu²¹⁹. The codon forIle²²⁰ was replaced with a TAA stop codon, and a new EcoRI site wasgenerated at the 3' end for cloning purposes. Amino acid 219 is the lastamino acid upstream of the predicted transmembrane domain, so thisconstruct encodes the whole extracellular domain of TF (referred to hereas rTF₁₋₂₁₉). The double-stranded oligonucleotide was also ligated tothe 5' end of the clone, but as it has no initiator ATG codon, it ispredicted to have no effect on expression of the recombinant protein.

The resulting 805 bp TF₁₋₂₁₉ cDNA was digested with EcoRI and subclonedinto the EcoRI site of the mammalian expression vector pCDNAI (availablefrom Invitrogen, San Diego, Calif.) in the proper orientation forexpression. The resulting construct was introduced into 293 cells byliposome-mediated transfection using Lipofectin Reagent (available fromGibco/BRL, Gaithersburg, Md., and used according to the manufacturer'sdirections). Stable clones were selected by co-transfection of theconstruct with the plasmid pMAM-neo (available from Clontech, SanFrancisco, Calif.) in a 10:1 molar ratio. Stably transfected cell linesexpressing truncated TF were selected initially by treating the cellswith the neomycin analog G418 (Geneticin from Gibco/BRL, Gaithersburg,Md.) at 400 μg/ml. Subsequently, culture supernatants of survivingdrug-resistant colonies were screened for high levels of expression oftruncated TF using a two-site, sandwich, enzyme-linked immunoassay basedon two MAbs described by Morrissey, et al., Thrombosis Research52:247-261 (1988). One stable, clonally-derived cell line was chosenthat secreted truncated TF at levels of up to 1 mg per liter per day inroller bottle culture.

For production of recombinant truncated TF, cells are typically grown toconfluence in roller bottles in Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% iron-supplemented calf serum (HyCloneLaboratories, Logan, Utah). Upon reaching confluence, the cultures areshifted to DMEM containing 2% serum, and culture medium is collectedevery four days thereafter.

The nucleotide and amino acid sequence of truncated tissue factor (tTF)is shown in FIG. 1 and in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.The truncated tissue factor protein lacks the predicted transmembraneand cytoplasmic domains of tissue factor. This version of the proteinretains cofactor activity, as reported by Ruf, et al., "The isolatedextracellular domain of tissue factor is functional on phospholipidsurfaces," Thrombosis Haemostasis 62:347 (abstract) (1989) and Ruf, etal., "Phospholipid-independent and--dependent interactions required fortissue factor receptor and cofactor function,"J Biol Chem 266:2158-2166(1991). However, it has now been discovered that this truncated form oftissue factor fails to support conversion of factor VII to VIIa,allowing it to be used in a specific clotting assay for factor VIIa freefrom interference by factor VII.

The essential difference between truncated tissue factor and wild-typetissue factor is that truncated tissue factor is no longer tethered tothe phospholipid membrane surface. It is therefore expected that othermethods for preparing truncated tissue factor can be used to generate asoluble form of tissue factor that retains some cofactor activity whileno longer stimulating conversion of factor VII to factor VIIa. Methodsinclude enzymatic digestion of wild-type tissue factor to separate thepredicted extracellular domain from the transmembrane region.Recombinant human TF is available from Calbiochem. Precise positioningof the stop codon following amino acid 219 is believed to not beessential to make functional truncated TF, and other placements of astop codon near amino acid 219 are predicted to yield an equivalentproduct with respect to its utility in the factor VIIa assay describedherein.

A factor VIIa standard curve must be generated for each new batch of tTFreagent, and the curve is applicable only for the assay configurationtested (e.g., for the specific manner of performing the manual tilt-tubemethod, or for the specific automated or semi-automated coagulationmeasuring device used to generate the standard curve). Changing from onemeasuring device to another, or changing batches of tTF reagent, willnecessitate generating a new standard curve.

3. Purified Proteins and Deficient Plasmas

Factor VII can be prepared as described by Fair, Daryl S., "Quantitationof Factor VII in the plasma of normal and Warfarin-treated individualsby radioimmunoassay," Blood 62:784-791 (1983). FVII can be converted tofVIIa by incubation of the purified protein with factor Xa immobilizedon Affi-Gel™ 15 beads (Bio-Rad). Conversion can be monitored bySDS-polyacrylamide gel electrophoresis of reduced samples. Free factorXa in the fVIIa preparation can be detected with the chromogenicsubstratemethoxycarbonyl-D-cyclohexylglycyl-glycyl-arginine-p-nitroanilideacetate (Spectrozyme™ FXa, American Diagnostica, Greenwich, Conn.) at0.2 mM final concentration in the presence of 50 mM EDTA. RecombinantfVIIa can also be purchased from Novo Biolabs (Danbury, Conn.).

In the preferred embodiment, test plasma consists of platelet-poor,citrated plasma, although other anticoagulants can be used such asethylenediamine tetracetic acid (EDTA) or sodium or potassium oxalate,and platelets do not have to be removed. This is prepared using standardhematologic techniques by drawing a small sample of blood byvenipuncture into a citrate anticoagulant (ACD or CPD), from whichplasma is prepared by centrifugation.

In the preferred embodiment, the reference plasmas from which thecalibration standard curve is generated consist of known quantities ofpurified, recombinant human factor VIIa added to human factorVII-deficient plasma. The human factor VII-deficient plasma can be fromcongenitally deficient donors (available from George King Biomedical,Overland Park, Kans.) or can be immunodepleted plasma (available fromAmerican Diagnostica, Greenwich, Conn.). Concentrations of added factorVIIa range from 1 pg/ml to 10 μg/ml. A log-log plot of clotting time vs.factor VIIa concentration is prepared and a curve (4th order polynomial)is fit using regression analysis. Clotting times for unknown plasmasamples are converted to factor VIIa concentrations using graphicalanalysis or a computer program.

Values obtained using the preferred embodiment of this assay for plasmalevels of factor VIIa range from 0.34 to 4.4 ng/ml.

The plasma must be handled and stored in containers made of materialsknown not to promote activation of the contact system of coagulation.This includes the materials the plasma comes in contact with during theassay. The manual assay typically employs tubes formed of plastics suchas polystyrene, polyethylene and polypropylene. An example of anunacceptable material is untreated glass, well-known for its ability toactivate the contact coagulation system. Glass treated bysiliconization, or by other methods known to eliminate contactactivation of plasma, are also acceptable.

4. Ionic Calcium

Typically, ionic calcium is provided as a 25 mM solution of CaCl₂.Calcium chloride is available from a number of commercial sources.

Assays for Activated Factor VII Using the tTF

The assay can be performed using the reagents described above andobtained individually, or as a kit to be used in clinical coagulationlaboratories, alone or in combination with other assays such asantigen-antibody assays. The essential components of the kit are thetruncated tissue factor reagent and a set of factor VIIa standardsconsisting of factor VII-deficient (or factor VII-depleted) human plasmasupplemented with known concentrations of factor VIIa. In most cases,the kit would be used with existing equipment such as coagulometers.Since the assay is conducted in a manner similar to that for the PTassay, minimal training of personnel is required.

The sensitivity of the tTF reagent to factor VIIa levels, expressed asthe decrease in clotting time of factor VIIa-containing plasma whenassayed in the presence versus the absence of tTF reagent, is a functionof the concentrations of both tTF and phospholipid vesicles (cephalin).Therefore, the sensitivity of the reagent can be increased, i.e., theclotting time at a given factor VIIa concentration shortened, byincreasing the concentration of tTF or cephalin in the reagent.Sensitivity of the reagent is adjusted by varying the concentrations oftTF or cephalin to achieve the desired range of clotting times relativeto the range of factor VIIa levels to be measured. Preferably, thecephalin concentration is kept constant at the strength normallyemployed for PTT or TGT assays, and the sensitivity of the tTF reagentis adjusted by varying only the concentration of tTF.

For example, high sensitivity tTF reagents can be prepared using rabbitbrain cephalin and between 100 nM and 1 μM tTF. As illustrated in FIG.2, the high sensitivity reagent is most useful for manual,semi-automated, or automated detection of factor VIIa levels in theanticipated normal range (approximately 0.1 to 10 ng factor VIIa/ml). Alow sensitivity tTF reagent can be prepared using rabbit brain cephalinand between 10 and 50 nM tTF. As illustrated in FIG. 3, the lowsensitivity reagent is most useful for manual, semi-automated, orautomated detection of factor VIIa levels in the therapeutic range, upto approximately 5 μg factor VIIa/ml.

1. Manual Clotting Assays

In the preferred embodiment, the manual assay is performed as follows:

1. Pre-warm 12×75 mm polystyrene test tubes to 37° C. in a water bath.

2. Add 0.1 ml truncated tissue factor reagent per tube and allow to warmto 37° C. for at least 3 min.

3. Add 0.1 ml test plasma, mix and warm for exactly 30 sec.

4. Add 0.1 ml pre-warmed (37° C.) 25 mM CaCl₂ solution, mix immediately,and determine clotting time from point of calcium addition using themanual tilt-tube method. The endpoint is when a visible clot is formedin the tube.

FIG. 4 is a graph of a factor VIIa standard curve generated by themanual tilt tube method employing tTF.

2. Automated Measuring Devices for Coagulation Testing

Automated and semi-automated devices for coagulation testing areavailable from a variety of suppliers. An example of a fully automated,photometric-based detection instrument suitable for the tTF assay forfactor VIIa is the ACL™ 300 Automated Coagulation Laboratory availablefrom Instrument Laboratory Division of Fisher Scientific Co. (Lexington,Mass.). In principle, any device capable of determining prothrombintimes can be used for the tTF assay for factor VIIa. This includesdevices that detect clots photometrically (coagulometers) or usingelectrodes (fibrometers). The devices should be run in PT mode, and thedata should be recorded in seconds. A variety of automated andsemi-automated coagulation testing devices suitable for use with the tTFassay for factor VIIa are available from Scientific Products Division ofBaxter Diagnostics, Inc. (McGaw Park, Ill.).

3. Application of tTF in Chromogenic Substrate Assays and Other AssayConfigurations

The tTF assay for plasma factor VIIa levels can be adapted for any assayconfiguration that employs TF or thromboplastin by substituting the tTFreagent for thromboplastin. The resulting assay will then be capable ofmeasuring factor VIIa levels independent of the level of inactivatedfactor VII. An example of such an adaptation is substitution of tTFreagent for thromboplastin in the chromogenic substrate assay (alsoknown as coupled amidolytic assay) for factor VII (FVIIam; see, forexample, Seligsohn et al., "Coupled amidolytic assay for Factor VII: itsuse with a clotting assay to determine the activity state of FactorVII," Blood 52:978-988 (1978). Another example is substitution of tTFreagent for thromboplastin in alternative PT assays such as thoseperformed on whole, citrated blood, for example, the Coumatrak™ ProtimeTest System marketed by Du Pont Company, Wilmington, Del.

4. tTF Assay for Factor VIIa Levels in Plasmas with Abnormal ProthrombinTimes

The tTF clotting assay for plasma factor VIIa depends on adequate levelsof factors II, V, X and fibrinogen. Deficiencies in any of thesefactors, or the presence of inhibitors of these factors in plasma, couldresult in inaccurate measurement of factor VIIa levels. Accordingly, thetTF assay for factor VIIa levels should be run in parallel with astandard PT assay. Plasmas that exhibit PT values outside the acceptablenormal range must be diluted ten-fold or one hundred-fold with factorVII deficient plasma and re-assayed. The resulting value for plasmafactor VIIa is then multiplied by the dilution factor to calculate thelevel of factor VIIa present in the original, undiluted plasma.

5. FVIIag Levels

FVIIag levels can be measured with a commercial ELISA method, asdescribed by Amiral, et al., Clinical Chemistry 30:1512 (1984) andavailable from Diagnostica Stago (Asnieres, France).

Applications of the Assay

The assay is designed for the measurement of plasma levels of factorVIIa in individuals, with primary application as a clinical diagnosticagent, particularly as a screening test in order to identify thoseindividuals with elevated levels of factor VIIa. Such individuals arethought to have increased risk of thrombotic disorders. Furthermore,there may be other associations between elevated factor VIIa levels anddisease that could be established with the use of this assay.

Another application for this assay is to monitor plasma factor VIIalevels in hemophiliacs and other individuals undergoing factor VIIatherapy. Recombinant factor VIIa therapy is currently in clinicaltrials.

1. Monitor Plasma Factor VIIa Levels in Patients Treated withRecombinant Factor VIIa

Clinical trials of recombinant factor VIIa therapy for bleedingdisorders are currently underway, but without a reliable and specificassay for measuring the resulting factor VIIa levels. The tTF assayprovides a means for setting dose and monitoring efficacy in thesepatients.

2. Monitor Plasma Factor VIIa Levels in Patients Taking OralAnticoagulants, Particularly Low-Dose Coumadin Therapy

Patients on low-dose coumadin therapy show little change in clottingtimes in standard clotting assays, making it difficult to determineefficacy of the dose. For patients who respond to oral anticoagulanttherapy with depressed factor VIIa levels, the tTF assay provides ameans of monitoring such patients for efficacy, adjusting dose, etc.

3. Identify Individuals in the General Population with Increased Risk ofIschemic Heart Disease, Stroke, or Other Thrombotic Disease

Recent studies have indicated a possible correlation between elevatedfactor VIIa levels and risk of such disorders as ischemic heart disease,as reported by de Sousa, et al., "Factor VII hyperactivity in acutemyocardial thrombosis. a relation to the coagulation activation,"Thrombosis Research, 51:165-173 (1988); Hoffman, et al., "Factor VIIactivity state in coronary artery disease," J Lab Clin Med 111:475-481(1988); Gordon, et al., J Lab Clin Med 109: 409-413 (1987); andMitropoulos, "Hypercoagulability and Factor VII inhypertriglyceridemia," Seminars in Thrombosis and Hemostasis, 14:246-252(1988).

4. Screen Women During Pregnancy to Identify Those with Increased Riskof Complications of Pregnancy

Factor VII coagulant activity normally rises during pregnancy. The assaymay therefore be of use in screening the blood of pregnant women, sincethere are suggestions of a correlation between excessively elevatedplasma factor VIIa levels and fetal growth retardation, as described byScarabin, et al., "Is Factor VII activation in pregnant women relevantto fetal growth retardation?," Thrombosis Research, 45:845-850 (1987).

5. Screen Women Taking Oral Contraceptives to identify Risk Group forThrombotic Complications

Since FVIIc normally rises in pregnancy, it may also rise during use oforal contraceptives. It is possible that elevation of plasma factor VIIacould be an underlying cause of the thrombotic episodes that occur insome women using oral contraceptives. Alternatively, elevated factorVIIa levels in these women may be a marker of a "prethrombotic state".In either case, it is of clinical value to identify reliably asubpopulation of women who experience an excessive rise in factor VIIalevels when taking oral contraceptives.

6. Identify Individuals with Undetected Malignancy

Fibrin deposition and other indicators of activation of the coagulationsystem are frequently associated with malignant tumors. Elevated plasmafactor VIIa levels may constitute a sensitive means of indicating thepresence of otherwise undetected tumors.

7. Identify and/or Monitor Patients with Inflammatory Diseases, Sepsis,and Incipient DIC

Activation of coagulation often accompanies inflammation, sepsis, andforms the basis of disseminated intravascular coagulation (DIC).Diagnosis of elevated plasma factor VIIa could serve as an early markerfor these conditions.

8. Assess Degree of Thrombotic Risk in Patients Who Have Diseases Thatare Known or Suspected to be Associated with Increased Tendency TowardThrombosis

Plasma factor VIIa levels serve as an extremely sensitive indicator foractivation of the clotting cascade in individuals; therefore, measuringfactor VIIa can be used to help assess the actual thrombotic risk inpatients who have diseases known to be associated with increasedtendency toward thrombosis. Measuring factor VIIa levels may also be ofvalue for monitoring effectiveness of anticoagulant therapy (orprophylaxis) in such individuals. Diseases with a known or suspectedrisk of thrombotic episodes include preeclampsia, hypertension, certainmalignancies, hyperlipidemia, hypercholesterolemia, protein Cdeficiency, and unexplained recurrent venous and arterial thrombosis. Inaddition, monitoring factor VIIa levels may be of use duringthrombolytic therapy of patients with acute myocardial infarction orstroke, and during and following vascular surgery, includingangioplasty.

Modifications and variations of the compositions for assaying factorVII, and methods of use thereof, will be obvious to those skilled in theart from the foregoing detailed description. Such modifications andvariations are intended to come within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..23                                                           (D) OTHER INFORMATION: /note= "Synthetic Oligonucleotide"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AATTTAGAGAATAAGAATTCGGG23                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: /note= "Synthetic Oligonucleotide"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CCCGAATTCTTATTCTCTA19                                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 795 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 1..33                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 34..786                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 130..786                                                        (D) OTHER INFORMATION: /product="Truncated Tissue Factor"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGTTCCGCTCGATCTCGCCGCCAACTGGTAGACATGGAGACCCCTGCCTGGCCC54                      MetGluThrProAlaTrpPro                                                         32-30                                                                         CGGGTCCCGCGCCCCGAGACCGCCGTCGCTCGGACGCTCCTGCTCGGC102                           ArgValProArgProGluThrAlaValAlaArgThrLeuLeuLeuGly                              25-20-15-10                                                                   TGGGTCTTCGCCCAGGTGGCCGGCGCTTCAGGCACTACAAATACTGTG150                           TrpValPheAlaGlnValAlaGlyAlaSerGlyThrThrAsnThrVal                              515                                                                           GCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTG198                           AlaAlaTyrAsnLeuThrTrpLysSerThrAsnPheLysThrIleLeu                              101520                                                                        GAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGC246                           GluTrpGluProLysProValAsnGlnValTyrThrValGlnIleSer                              253035                                                                        ACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACA294                           ThrLysSerGlyAspTrpLysSerLysCysPheTyrThrThrAspThr                              40455055                                                                      GAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTAC342                           GluCysAspLeuThrAspGluIleValLysAspValLysGlnThrTyr                              606570                                                                        TTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGT390                           LeuAlaArgValPheSerTyrProAlaGlyAsnValGluSerThrGly                              758085                                                                        TCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTAC438                           SerAlaGlyGluProLeuTyrGluAsnSerProGluPheThrProTyr                              9095100                                                                       CTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTG486                           LeuGluThrAsnLeuGlyGlnProThrIleGlnSerPheGluGlnVal                              105110115                                                                     GGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGA534                           GlyThrLysValAsnValThrValGluAspGluArgThrLeuValArg                              120125130135                                                                  AGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTA582                           ArgAsnAsnThrPheLeuSerLeuArgAspValPheGlyLysAspLeu                              140145150                                                                     ATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACA630                           IleTyrThrLeuTyrTyrTrpLysSerSerSerSerGlyLysLysThr                              155160165                                                                     GCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAA678                           AlaLysThrAsnThrAsnGluPheLeuIleAspValAspLysGlyGlu                              170175180                                                                     AACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAAC726                           AsnTyrCysPheSerValGlnAlaValIleProSerArgThrValAsn                              185190195                                                                     CGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGG774                           ArgLysSerThrAspSerProValGluCysMetGlyGlnGluLysGly                              200205210215                                                                  GAATTTAGAGAATAAGAATTC795                                                      GluPheArgGlu                                                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 251 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetGluThrProAlaTrpProArgValProArgProGluThrAlaVal                              32-30-25-20                                                                   AlaArgThrLeuLeuLeuGlyTrpValPheAlaGlnValAlaGlyAla                              15-10-5                                                                       SerGlyThrThrAsnThrValAlaAlaTyrAsnLeuThrTrpLysSer                              151015                                                                        ThrAsnPheLysThrIleLeuGluTrpGluProLysProValAsnGln                              202530                                                                        ValTyrThrValGlnIleSerThrLysSerGlyAspTrpLysSerLys                              354045                                                                        CysPheTyrThrThrAspThrGluCysAspLeuThrAspGluIleVal                              505560                                                                        LysAspValLysGlnThrTyrLeuAlaArgValPheSerTyrProAla                              65707580                                                                      GlyAsnValGluSerThrGlySerAlaGlyGluProLeuTyrGluAsn                              859095                                                                        SerProGluPheThrProTyrLeuGluThrAsnLeuGlyGlnProThr                              100105110                                                                     IleGlnSerPheGluGlnValGlyThrLysValAsnValThrValGlu                              115120125                                                                     AspGluArgThrLeuValArgArgAsnAsnThrPheLeuSerLeuArg                              130135140                                                                     AspValPheGlyLysAspLeuIleTyrThrLeuTyrTyrTrpLysSer                              145150155160                                                                  SerSerSerGlyLysLysThrAlaLysThrAsnThrAsnGluPheLeu                              165170175                                                                     IleAspValAspLysGlyGluAsnTyrCysPheSerValGlnAlaVal                              180185190                                                                     IleProSerArgThrValAsnArgLysSerThrAspSerProValGlu                              195200205                                                                     CysMetGlyGlnGluLysGlyGluPheArgGlu                                             210215                                                                        __________________________________________________________________________

I claim:
 1. A method for measuring the concentration of coagulationfactor VIIa in a patient's plasma sample, comprising the steps of:(a)mixing a quantity of a plasma sample with a soluble truncated tissuefactor, said soluble truncated tissue factor characterized by itsinability to bind to phospholipids and its inability to supportconversion of factor VII to factor VIIa, to form a first mixture; (b)thereafter, adding an effective amount of calcium ions to said firstmixture to form a second mixture and to initiate clotting of said secondmixture; (c) determining a plasma sample clotting time for said secondmixture measured from a time of addition of said calcium ions to a timeof clot formation; and (d) comparing said plasma sample clotting time toa standards clotting time determined for factor VIIa of a knownconcentration.
 2. The method of claim 1, wherein said standards clottingtime is determined for a plurality of known concentrations of factorVIIa diluted in factor VII-deficient plasma by adding a like quantity ofsaid soluble truncated tissue factor and said calcium ions as that addedto said plasma sample to said known concentrations of factor VIIadiluted in factor VII-deficient plasma and measuring said standardsclotting time from a time of addition of said calcium ions to a time ofclotting, and correlating said known concentrations of factor VIIa withsaid standards clotting time to form a standard curve.
 3. The method ofclaim 1 or 2, wherein said soluble truncated tissue factor is theproduct of enzymatic digestion of wild-type tissue factor to separatethe predicted extracellular domain from the transmembrane region.
 4. Themethod of claim 1 or 2, wherein said truncated tissue factor is theproduct of recombinant expression of the DNA sequence of SEQ ID NO: 3,wherein the stop codon is positioned near the codon for amino acid 219.5. The method of claim 1, wherein said plasma sample is diluted betweenabout 1:10 and 1:100 with factor VII-deficient plasma prior to step (a).6. A method for measuring the concentration of coagulation factor VIIain a patient's plasma sample, comprising the steps of:(a) mixing aquantity of a plasma sample with a combination of a soluble truncatedtissue factor and a phospholipid agent, said soluble truncated tissuefactor characterized by its inability to bind to phospholipids and itsinability to support conversion of factor VII to factor VIIa, to form afirst mixture; (b) thereafter, adding an effective amount of calciumions to said first mixture to form a second mixture and to initiateclotting of said second mixture; (c) thereafter, determining a plasmasample clotting time for said second mixture measured from a time ofaddition of said calcium ions to a time of clot formation; and (d)comparing said plasma sample clotting time to a standards clotting timedetermined for factor VIIa of a known concentration.
 7. The method ofclaim 6, wherein said standards clotting time is determined for aplurality of known concentrations of factor VIIa diluted in factorVII-deficient plasma by adding a like quantity of said soluble truncatedtissue factor and said calcium ions as that added to said plasma sampleto said known concentrations of factor VIIa diluted in factorVII-deficient plasma and measuring said standards clotting time from atime of addition of said calcium ions to a time of clotting, andcorrelating said known concentrations of factor VIIa with said standardsclotting time to form a standard curve.
 8. The method of claim 6 or 7,wherein said soluble truncated tissue factor is the product of enzymaticdigestion of wild-type tissue factor to separate the predictedextracellular domain from the transmembrane region.
 9. The method ofclaim 6 or 7, wherein said truncated tissue factor is the product ofrecombinant expression of the DNA sequence of SEQ ID NO: 3, wherein thestop codon is positioned near the codon for amino acid
 219. 10. Themethod of claim 6, wherein said phospholipid agent is added in an amountof about 80 μg per milliliter of said first mixture.
 11. The method ofclaim 6, wherein said plasma sample is diluted between about 1:10 and1:100 with factor VII-deficient plasma prior to step (a).
 12. A methodfor measuring the concentration of coagulation factor VIIa in apatient's plasma sample, comprising the steps of:(a) mixing a quantityof a plasma sample with a reagent to form a first mixture, said reagentcomprising a soluble truncated tissue factor and an effective amount ofcalcium ions to initiate clotting of said first mixture, said solubletruncated tissue factor characterized by its inability to bind tophospholipids and its inability to support conversion of factor VII tofactor VIIa; (b) determining a plasma sample clotting time for saidfirst mixture measured from a time of addition of said reagent to a timeof clot formation; and (c) comparing said plasma sample clotting time toa standards clotting time determined for factor VIIa of a knownconcentration.
 13. The method of claim 12, wherein said standardsclotting time is determined for a plurality of known concentrations offactor VIIa diluted in factor VII-deficient plasma by adding a likequantity of said reagent as that added to said plasma sample to saidknown concentrations of factor VIIa diluted in factor VII-deficientplasma and measuring said standards clotting time from a time ofaddition of said reagent to a time of clotting, and correlating saidknown concentrations of factor VIIa with said standards clotting time toform a standard curve.
 14. The method of claim 12, wherein said reagentfurther comprises a phospholipid agent.
 15. The method of claim 13,wherein said reagent further comprises a phospholipid agent.
 16. Themethod of claim 12 or 13, wherein said soluble truncated tissue factoris the product of enzymatic digestion of wild-type tissue factor toseparate the predicted extracellular domain from the transmembraneregion.
 17. The method of claim 12 or 13, wherein said truncated tissuefactor is the product of recombinant expression of the DNA sequence ofSEQ ID NO: 3, wherein the stop codon is positioned near the codon foramino acid
 219. 18. The method of claim 12, wherein said plasma sampleis diluted between about 1:10 and 1:100 with factor VII-deficient plasmaprior to step (a).